There are many different types of cancers and means of treating the cancers. The most typical way of treating cancer is through the use of chemotherapeutic agents that selectively target the more rapidly proliferating tumor cells relative to the host cells. These have serious side effects, including death of normal cells, mucositis, nausea, and may give rise to drug resistant tumor cells. Antibodies and antibody targeted therapeutics are a desirable alternative to chemotherapeutics since they are more selective, but are limited to the few tumors expressing antigens that can be effectively targeted. Drugs that inhibit angiogenesis are another approach having fewer side effects, but have been found to only limit tumor growth, not kill existing tumors.
Viruses are an alternative approach for combating cancer. One approach to treating tumors is to use replication-incompetent viral vectors to deliver genes to the tumor. Replication-incompetent viruses have value in delivering genes to the area of the tumor that might then alter the general environment near the tumor, making it less hospitable to the tumor. There are a number of interesting strategies based on viral vectors, including delivery of suicide genes such as HSV-tk or ricin, genes that reduce vascular proliferation, restore cell cycle control and encourage apoptosis such as p53, or stimulate an immune response (IL-12, interferon, tumor necrosis factor). One problem with replication incompetent viruses is that, even with direct injection into a tumor, the number of cells infected in a solid tumor is generally quite small, averaging about 7% of the total tumor cell population (Puumalainen ei al., Adv Exp Med Biol, 451:505-509 (1998); Rainov and Ren, Acta Neurochir Suppl, 88:113-123 (2003)). Thus with viruses that do not replicate, it has not been possible to infect a substantial percentage of the tumor cells.
A primary advantage of conditional replication competent viruses is that they infect and kill tumor cells, and then their new viral progeny are released to kill additional tumor cells. In contrast to replication incompetent viral vectors that only infect a small number of tumor cells, a small injection of replication competent VSV or Sindbis virus can lead to a rapid and complete intratumoral spread of the virus. Beneficially, due to local self-amplification, replication competent viruses may be effective with one single application, whereas repetitive injections have to be applied for the less effective replication incompetent agents. A number of viruses have been shown to have oncolytic potential, including recombinant herpes, adeno, polio and alpha viruses. Myxoma virus was effective in killing medulloblastoma, and infection was increased in combination with Rapamycin (Lun, et al., Cancer Res., 67(18):8818-8827 (2007). Herpes simplex virus has been rendered conditionally replication-competent for tumors by inserting mutations in several HSV genes. Oncolytic adenovirus agents specifically restrict their replication to p53 deficient tumor cells, and retrovirus to tumors with activated Ras-signaling pathways (Chiocca, Nat. Rev. Cancer, 2: 938-950 (2002); Rainov and Ren, Acta Neurochir Suppl, 88:113-123 (2003)). These strategies are restricted to defined mutations of the tumor. Oncolytic viruses that are not strictly dependent on a single gene mutation are therefore desirable to treat cancers that exhibit a heterogenous array of genetic aberrations.
One replication competent virus with oncolytic potential is vesicular stomatitis virus (VSV) (Lun, et al. J. Nat. Can. Instit., 98(21): 1546-1557 (2006), Wu, et al., Clin. Cancer Res., 14(4):1218-1227 (2008), Ozduman, et al., J. Virol., 83(22):11540-11549 (2009), Wollmann, et al., J. Virol, (2009 Epub; 2010 J. Virology 84(3). VSV is an RNA virus that uses a negative strand RNA to encode its genome. Although some viruses may express over a hundred different genes, VSV with its 11-12 kilobase genome encodes five structural genes, N, P, M, G, and L, encoding proteins that each may have multiple functions. The virus envelope consists of a host cell-derived lipid bilayer that contains several hundred copies of viral glycoprotein spikes (G-protein) and forms a characteristic bullet shape. The nucleocapsid protein N forms a tight complex with the helical coiled non-segmented RNA genome and the viral matrix protein M bridges the gap between nucleocapsid and viral envelope. A complex formed by the phosphoprotein P and the large polymerase L facilitates viral polymerase activity. Phosphorylation is thought to play a role in the function of the of P protein, for example, unphosphorylated P is much less active in supporting viral transcription at low concentrations (Spadafora et al., J. Virol., 70(7): 45-38-4548 (1996)).
VSV binds to the cell membrane, becomes internalized, and then replicates in the cell cytoplasm. VSV replicates fairly quickly, about 6 hours for progeny virus to bud from the plasma membrane of the infected cell. A single infected cell can release many VSV progeny, although some of the progeny are defective particles. VSV blocks the ability of infected cells to transport mRNA out of the nucleus (VSV M-protein blocks nuclear pores), and usurps the host cell translational mechanisms to synthesize primarily viral gene products. VSV is found around the world. Many mammals, including humans, can be infected by the virus which is probably spread by biting flies (de Mattos et al. In Fields of Virology, pp. 1245-1277, (Knipe, et al., Eds) (2001)). Humans show only modest symptoms, or none, with infection which clears as the immune system eliminates the virus. In some rural regions of central America, the majority of adults show seropositivity with antisera against VSV with few symptoms detectable (Tesh, et al., Am. J. Epidemiol., 90:255-261, (1969)). Replication competent VSVs are in human clinical trials as a carrier virus to generate immune responses against more dangerous viruses, including HIV, influenza, and papilloma viruses cloned into the VSV genome (Roberts, et al., J. Virol., 73:3723-32 (1999); Roberts, et al., J. Virol., 78: 3196-3199 (2004); Okuma et al., J. Virol. 346(1):86-97 (2006); Rose et al., Cell, 106:539-549 (2001)).
VSV has demonstrated potential as an oncolytic virus. In a battery of in vitro and in vivo tests, VSV showed the greatest promise among nine replication competent viruses including Sindbis, human and mouse cytomegalovirus, pseudorabies virus, the parvoviruses MVMi and MVMp, and others that have been described as having oncolytic potential (Wollmann et al, J. Virol., 79(10): 6005-6022 (2005)). Normal cells possess innate mechanisms to reduce or block viral infections. VSV replication can be attenuated or blocked by cellular interferon (IFN). Defects in this system are thought to contribute to the oncolytic activity of VSV (Wollmann, et al., J. Virol., 81(3):1479-1491 (2007)). In addition to destroying glioblastoma cells, VSV has also shown promise outside the brain as an anti-cancer agent in recent experimental studies on various solid tumors, for example carcinoma of colon (Stojdl et al., Cancer Cell, 4:263-275 (2003)), breast (Ebert et al., Cancer Gene Ther, 12:350-358 (2005)), prostate (Ahmed et al., Virology, 330:34-49 (2004)) and liver (Shinozaki et al, 2005) or hematological malignancies such as leukemia (Lichty et al., 2004b).
To further enhance the potential selectivity of VSVs for brain cancer cells, a recombinant wildtype-based VSV expressing a gene coding for green fluorescent protein (GFP), VSV-G/GFP (7,30), was grown for many generations on human glioblastoma cancer cells using a protocol that enhanced viral fitness based on viral internalization, replication, and cell selectivity. This cell-adapted propagation resulted in a virus that displayed an enhanced rate of infection and replication on the U-87MG glioblastoma line used to grow the virus and was named VSV-rp30 (Wollmann et al, J. Virol., 79(10): 6005-6022 (2005), Ozduman, et al., J. of Neurosci., 28(8): 1882-1893 (2008)). Identification of the differences between VSV-rp30 and less effective VSV viruses, such as the parent strain, would provide an avenue for development of a range of oncolytic viruses with improved oncolytic potential for anti-cancer therapeutics.
It is an object of the invention to provide isolated oncolytic VSV virus with enhanced oncolytic potential, compared to naturally occurring wild strains.
It is a further object of the invention to provide mutant VSVs with an increased replicative capacity in cancer cells relative to normal cells.
It is another object of the invention to provide a viral composition effective to reduce tumor burden.
It is another object of the invention to provide methods for making and using the viral compositions for treating cancer.